Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Modulation of Ca2+ oscillation and apamin-sensitive,Ca2+-activated K+ current in rat gonadotropes

In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca²⁺ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃ ], which in turn induces an oscillatory activation of apamin-sensitive Ca²⁺-activated K⁺ current, I _K(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) and I _K(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca²⁺]_i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4-phorbol 12,13-didecanoate (4-PDD), reduced the frequency of GnRH-induced [Ca²⁺]_i oscillation and augmented the I _K(Ca) induced by any given level of [Ca²⁺]_i. The slowing of oscillations and the enhancement of I _K(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5-O-(3-thio-triphosphate) (GTP-[S]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P ₃, phorbol esters enhanced I _K(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced I _K(Ca) without affecting [Ca²⁺]_i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca²⁺]_i oscillations probably by actions on phospholipase C, and it enhances I _K(Ca) probably by a direct action on the channels.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Divalent cations activate small- (SK) and large-conductance (BK) channels in mouse neuroblastoma cells: selective activation of SK channels by cadmium

Effects of Cd²⁺, Co²⁺, Fe²⁺ and Mg²⁺ (1 M and 100 M) and Pb²⁺ (1 M and 90 M) on single-channel properties of the small-conductance (SK) and large-conductance (BK) Ca²⁺-activated K⁺ channels were investigated in inside-out patches of N1E-115 mouse neuroblastoma cells. Cd²⁺, Co²⁺ and Pb²⁺, but not Fe²⁺ and Mg²⁺, cause SK channel opening. The potency of the metals in enhancing the SK channel-open probability follows the sequence Cd²⁺ Pb²⁺>Ca²⁺>Co²⁺ Mg²⁺, Fe²⁺. The four metals that cause SK channel opening are equipotent in enhancing the opening frequency of SK channels. The BK channel is activated by Pb²⁺ and Co²⁺, whereas Cd²⁺, Fe²⁺ and Mg²⁺ are ineffective. The potency of the metals in enhancing BK channel-open probability, open time and opening frequency follows the sequence Pb²⁺>Ca²⁺>Co²⁺Cd²⁺, Mg²⁺, Fe²⁺. The results show that SK channels are much more sensitive to Cd²⁺ than BK channels and indicate that Cd²⁺ is a selective agonist of SK channels. It is concluded that the various metal ions bind to the same regulatory site(s) at which Ca²⁺ activates the SK and BK channels under physiological conditions. The different potency sequences of metal ions with respect to BK and SK channel activation indicate that the regulatory sites of these Ca²⁺-activated K⁺ channeles have distinct chemical and physical properties.     

Ca2+ release in cultured rat epididymal cells during hypoosmotic swelling

Microfluorimetric studies were carried out to investigate the effects of hypoosmotic swelling on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single rat epididymal cells. In Ca²⁺-free solution containing 50 mol/l ethylenebis(oxonitrilo)tetraacetate (EGTA) hypoosmotic swelling (–160 mosmol/l) induced a transient rise in [Ca²⁺]_i which was either monophasic, biphasic or oscillatory. The [Ca²⁺]_i responses to repeated hypoosmotic stimulations followed a decremental pattern. However, if 2.5 mmol/l Ca²⁺ was admitted during the recovery period between successive stimulations, the second and the third [Ca²⁺]_i responses were slightly greater than the first. Increasing the change in osmolarity from –14±1.0 to –154±1.5 mosmol/l increased the rise in [Ca²⁺]_i but reduced the [Ca²⁺]_i response to subsequent ionomycin stimulation (4 mol/l). The swelling- and the ionomycin-induced rises in [Ca²⁺]_i followed a reciprocal pattern. It was suggested that intracellular Ca²⁺ release in response to cell swelling in the epididymal epithelium might play a role in cell volume regulation and the control of epididymal fluid osmolarity.     

Activation of K+ and Cl− channels by Ca2+ and cyclic AMP in dissociated kidney epithelial (MDCK) cells

In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl^– (5 mM) but had little effect in high Cl^– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl^– channels. The Ca²⁺-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl^– solutions, consistent with K⁺ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca²⁺]_i as measured with the intracellular Ca²⁺ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl^– channels. However only l-epinephrine produced a [Ca²⁺]_i rise and a transient hyperpolarization (due to K⁺ channel opening), which preceeded the depolarization due to Cl^– channel opening. The l-epinephrine-induced [Ca²⁺]_i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca²⁺. In the latter only the slow secondary phase of the [Ca²⁺]_i rise was affected by Ca²⁺ removal. It is concluded that l-epinephrine activates K⁺ and Cl^– channels in a sequential manner in MDCK cells by Ca²⁺ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells Madin Darby Canine Kidney cells - [Ca²⁺]_i intracellular calcium concentration - [Cl^–]_i intracellular chloride concentration - [Cl^–]_o extracellular chloride concentration - [Na⁺+K⁺]_i intracellular concentration of Na⁺ and K⁺ - [Na⁺+K⁺]_o extracellular concentration of Na⁺ and K⁺ - E_M transmembrane potential - E_Cl chloride equilibrium potential - E_K potassium equilibrium potential - bis-oxonol [bis(1,3-diethylthio-barbiturate)] trimethine oxonol - DMSO dimethylsulfoxide - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid - NMG⁺ N-methylglucamine - RPMI medium Rosewell Park Memorial Institute medium     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Characterization of the high-conductance Ca2+-activated K+ channel in adult human skeletal muscle

Ca²⁺-activated K⁺ channels of a large conductance (BK_Ca) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BK_Ca conductances of 230 pS for symmetrical and 130 pS for physiological K⁺ distributions. Open probability increased with membrane depolarization and increasing internal [Ca²⁺]. The Hill coefficient was 2.0, indicating that at least two Ca²⁺ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5–20 s, was observed following large depolarizations, when extracellular K⁺ was lowered to physiological values. BK_Ca were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC₅₀ approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC₅₀ approx. 5 g/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC₅₀ <10 nM). Measurements on resealed fibre segments at physiological [K⁺] were in accordance with the single-channel data: only when intracellular [Ca²⁺] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K⁺ conductance with depolarizing voltage steps.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle

The effects of the stable thromboxane analogue U46619, the ₁-adrenergic agent phenylephrine and depolarization with high K⁺ on cytoplasmic Ca²⁺ ([Ca²⁺]_i) and force development were determined in rabbit pulmonary artery smooth muscle. Following stimulation with each of the excitatory agents, the time course of the [Ca²⁺]_i/force relationship described counter-clockwise hysteresis loops with the rise and fall in [Ca²⁺]_i leading, respectively, contraction and relaxation. The rank order of the force/[Ca²⁺]_i ratios evoked by the different methods of stimulation was: U46619 > phenylephrine high K⁺. The difference between the actions of U46619 and phenylephrine was due to the lesser Ca²⁺-releasing and greater Ca²⁺-sensitizing action of U46619. Both U46619 and phenylephrine also released intracellular Ca²⁺ in intact (non-permeabilized) preparations. The effects of the two agonists on force, at constant free cytoplasmic [Ca²⁺] maintained with EGTA, were also determined in preparations permeabilized with staphylococcal -toxin, in which intracellularly stored Ca²⁺ was eliminated with A23187. Sensitization of the contractile response to Ca²⁺ by agonists was indicated by the contractile responses of permeabilized muscles to U46619 and to phenylephrine, in the presence of constant, highly buffered [Ca²⁺]_i. These contractions were inhibited by GDP[S] and could also be elicited by GTP. We conclude that, in addition to changing [Ca²⁺]_i, pharmacomechanical coupling can also modulate contraction by altering the sensitivity of the regulatory/contractile apparatus of smooth muscle to [Ca²⁺]_i, through a G-protein-coupled mechanism.     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca²⁺ ([Ca²⁺]_i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca²⁺]_i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca²⁺]_i in a dose-dependent manner. [Ca²⁺]_i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10^–6M, and to 766±162 nmol/l with AVP, 10^–7 M. The increment in [Ca²⁺]_i by both hormones was by intracellular Ca²⁺ release and entry through plasma membrane Ca²⁺ channels. 8-Bromoadenosine-3, 5-cyclic monophosphate (8-BrcAMP), 10^–4M, increased [Ca²⁺]_i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 mol), ([Ca²⁺]_i changes: 86±4 to 390±29 nmol/l) and removal of bath Ca _o ²⁺ , ([Ca²⁺]_ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca²⁺]_i. Thapsigargin, 1.5 mol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca²⁺ transients, and partially inhibited PTH-mediated Ca²⁺ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca²⁺ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca²⁺]_i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca²⁺ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca²⁺ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca²⁺ and be modulated by different pathways which may have diverse effects on cTAL function.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca²⁺ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca²⁺ mobilization were investigated by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using Fura 2 in Ca²⁺-containing or Ca²⁺-free solution. The resting [Ca²⁺]_i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca²⁺, 10 M ACh or 128 mM K⁺ produced a phasic, followed by a tonic, increase in [Ca²⁺]_i but 20 mM caffeine produced only a phasic increase. In Ca²⁺-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca²⁺]_i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca²⁺. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 M ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca²⁺]_i following application of 20 mM caffeine. The level of [Ca²⁺]_i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca²⁺-free solution. These results suggest that both ACh and caffeine release Ca²⁺ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca²⁺]_i and inhibited the rate of decline of [Ca²⁺]_i following wasthout of high K⁺ or ACh in Ca²⁺-containing solution suggests that SR may negatively regulate the resting [Ca²⁺]_i in smooth muscle cells of the porcine coronary artery.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.